PROJECT 3: Type III secretion (TTS) represents one of the most complex mechanisms of protein translocation in biology. It also provides a striking example of the ability of pathogenic bacteria to manipulate their eukaryotic hosts. Since TTS control systems are almost always coupled to global regulatory factors that control virulence, understanding how TTS is regulated provides an expedient means to identify previously unknown determinants of pathogenesis. The goals of this project are to conduct a comprehensive assessment of the role in pathogenesis of TTS by B. pseudomallei and to use an understanding of the regulation of TTS to identify additional, unlinked virulence factors. Two aims are proposed: Aim 1. Characterize the relative roles of TTS1, TTS2 and TTS3 in B. pseudomallei-bost interactions. Deletion mutagenesis will be used to inactivate each TTSS individually and in combination. An array of assays will be used to evaluate the effects of mutational alterations on host-cell interactions in vitro and the establishment of infection, persistence, immunity and disease in murine models of infection. Aim 2. Characterize the regulatory mechanisms that control the expression of TTS loci and identify novel determinants of virulence. We will determine how TTS gene clusters are regulated and we will identify global regulatory factors with the potential to control multiple virulence determinants. Phenotypes resulting from mutations in regulatory loci will be evaluated in vitro and in vivo. We will also use an understanding of local and global regulatory circuitry to identify TTS effector molecules and co-regulated loci with the potential to encode novel determinants of virulence.